The invention is a process that reduces, eliminates or inactivates the agent that causes transmissible spongiform encephalopathy (TSE) from lipoproteins in a manner that does not adversely affect the biological activity of the lipoproteins.
Water-insoluble lipids like cholesteryl esters, triglycerides and the more polar phospholipids and unesterified cholesterol must travel through the aqueous environment of plasma (Bradely, W. A. and Gotto, A. M.: American Physiological Society, Bethesda, Md., pp 117-137 (1978)). The solubility of these lipids is achieved through physical association with proteins termed apolipoproteins, and the lipid-protein complexes are called lipoproteins (Dolphin, P. J., Can. J. Biochem. Cell. Biol. 63, 850-869 (1985)). Five distinct classes of lipoproteins have been isolated from human plasma: chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL) and lipoprotein (a) (Lp(a)) (Alaupovic, P. (1980) In Handbook of Electrophoresis. Vol. 1, pp. 27-46; Havel, R. J., Eder, H. A.; Bragdon, J. H., J. Clin. Invest. 34, 1343 (1955)). Dietary triglycerides and cholesterol are assembled by enterocytes (intestinal cells) into a chylomicron particle, which enters circulation through the lymphatic system (Brown, M. S. and Goldstein, J. L Sci. American (1984) 251, 58-66). Chylomicrons provide fatty acids to peripheral cells and cholesterol to liver. The liver in turn repackages cholesterol together with triglycerides into another lipoprotein called VLDL.
The function of VLDL is similar to chylomicrons, i.e. supply of free fatty acids to the muscle and adipose tissues and cholesterol to peripheral cells (Brown, M. S. and Goldstein, J. L Sci. American (1984) 251, 58-66). In the circulatory system, triglycerides in the VLDL particle are hydrolyzed by an enzyme called lipoprotein lipase (LPL) and additional processing by hepatic lipase finally converts it to LDL (Dolphin, P. J.: Can. J. Biochem. Cell. Biol. (1985) 63, 850-869). Thus, the liver produces VLDL, the precursor of LDL. Because VLDL is a precursor to LDL, decreases in VLDL production translate into lowered LDL levels. High levels of circulating LDL have been positively correlated with the development of coronary disease. While LDL cholesterol is clearly an independent positive risk factor, HDL cholesterol is considered to be a negative risk factor (Tribble, D. L.; Krauss, R. M. Advances in Internal Medicine (1993) 38:1-29).
HDL promotes reverse cholesterol transport, a process by which excess cholesterol is extracted from peripheral cells by HDL and delivered to the liver for its elimination. Reverse cholesterol transport, therefore, reduces cholesterol accumulation in the artery wall (Reichl, D and Miller, N. E., Arteriosclerosis 9, 785 (1989)). Because there is no cholesterol accumulation in extrahepatic organs, cholesterol must be transported to the liver by HDL for ultimate excretion into bile, either as free cholesterol, or as bile acids that are formed from cholesterol (Kwiterovich, P. O., Amer. J. Cardiol. 82, 13Q, (1998)). HDL can acquire part of its anti-atherogenic character by promoting the reverse transport of cholesterol.
In humans, low HDL cholesterol levels can relate to defects in synthesis or catabolism of Apo-Al, with catabolic defects being more common (Brinton, E. A., et al., Ateriosclerosis Thromb. 14, 707 (1994)); Fridge, N., et al., Metabolism 29, 643 (1980)). Low HDL is often associated with hypertriglyceridemia, obesity, and insulin resistance (Brinton, E. A., et al., Ateriosclerosis Thromb. 14, 707 (1994)). HDL from hypertriglyceridemic subjects characterized by low HDL levels have small HDL particles which are susceptible to renal filtration and degradation. The liver is the principal organ of HDL apolipoprotein degradation (Horowitz, B. S., et al., J Clin. Invest. 91, 1743 (1993)).
HDL has other important characteristics that can contribute to its anti-atherogenic properties. Recent evidence suggests that HDL can have antioxidant and antithrombotic properties (Tribble, D., et al., J. Lipid Res. 36, 2580 (1995); Mackness, M. I., et al., Biochem. J. 294, 829 (1993); Zeither, A. M., et al., Circulation 89, 2525 (1994)). HDL can also affect the production of some cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), (Cockerill, G. W., et al., Arterioscler. Thromb., 15, 1987 (1995)). These properties of HDL also provide protection against coronary artery disease.
Cholesterol and cholesterol-containing lipoproteins obtained from mammalian serum are also useful to promote the growth of various organisms. J. Bacteriol., Vol. 135, pp. 818-827 (1978) describes the use of a cholesterol-containing bovine serum fraction in the growth of Mycoplasma pneumoniae and Mycoplasma arthritidis. J. Gen. Microbiology, Vol. 116, pp. 539-543 (1980) describes the use of USP cholesterol in the growth of Treponema hyodysenteriae.
U.S. Pat. No. 4,290,774 describes the production of a specific cholesterol-rich fraction from mammalian plasma or serum by a process that involves the step of treatment with an alkaline carbonate and an alkaline earth salt. Zeit. Klin. Chem. 6(3), pp. 186-190 (1968) describes the removal of certain lipoproteins from human serum by use of colloidal silicic acid.
U.S. Pat. No. 4,762,792 discloses a process for isolating a cholesterol-rich fraction from mammalian blood plasma or serum using a silica adsorbant followed by several alkaline steps.
The transmissible degenerative encephalopathies, also known as spongiform encephalopathies or transmissible spongiform encephalopathies (TSE), constitute a distinct group of fatal neurological diseases of mammals. In 1994, Prusiner described the xe2x80x9cremarkable discoveries in the past three decades (which have) led to the molecular and genetic characterization of the transmissible pathogen causing scrapie in animals and several illnesses in humans: Kura, Creutzfeldt Jacob Disease (CJD), Gerstmann-Straussler-Scheinker disease, and Fatal Familial Insomnia. To distinguish this infectious pathogen from viruses and viroids, the term PRION was introduced to emphasize its proteinaceous and infectious nature.xe2x80x9d Prusiner, xe2x80x9cBiology and Genetics of Prion Diseases,xe2x80x9d Annu. Rev. Microbiol. 48:655-686 (1994). A prion is composed solely of one host-encoded protein (referred to as the xe2x80x9cPrPxe2x80x9d) that resists most inactivation procedures such as heat, radiation and/or proteases. The latter characteristic has led to the term xe2x80x9cprotease-resistant isoformxe2x80x9d of the prion protein. Amino acid sequences of PrP obtained from normal and infected brains are identical, and these two proteins differ only in their biochemical and biophysical behavior. In particular, PrP from TSE individuals resists proteinase K digestion, while PrP-c found in normal individuals is degraded by proteinase K. PrP-res is not associated with an increase in mRNA, instead the accumulation is post-translational. Infected animals accumulate their own PrP-res, not that used for, or which created, the infection (Dormont, Agents that cause transmissible subacute spongiform encephalopathies, Biomed and Pharmacother 1999:53:3-8).
Prions resist almost all of the general procedures used to inactivate conventional viruses. Dormont, id.
Mad cow disease, or bovine spongiform encephalopathy (BSE), is the most well known TSE. All of the transmissible spongiform encephalopathies are progressive degenerative disorders that affect the central nervous system of animals and humans. These neurologic diseases take part of their name from the spongiform or xe2x80x9csponge-likexe2x80x9d degeneration of brain tissue that they cause. These diseases share many clinical and pathological features, and some scientific evidence now suggests that they develop through common or closely related mechanisms. This degeneration is most common in the cerebral cortex, the basal ganglia and the thalamus. Among other unique features, all of these diseases are associated with the accumulation of an abnormal form of the prion protein in nerve cells that eventually leads to the death of the host.
These diseases can be transmitted from one host to another much like an infection; unlike more typical forms of infectious diseases; however, the transmissible spongiform encephalopathies have incubation periods that are measured in intervals of months to many years. While prion diseases can all be transmitted from one host to another, it remains unknown whether a virus-like infectious agent or the abnormal prion protein itself (prion) causes the conversion of normal to abnormal protein.
In some cases, transmissible spongiform encephalopathies develop through genetic mutations and therefore occur as familial or hereditary disorders. Regardless of the cause (sporadic, infectious or familial), once the disease has manifested itself, it typically progresses over a period of months and inevitably leads to the death of the affected host. Questions about how these diseases are transmitted and whether they can cross species barriers remain only partially answered. Evidence does exist that BSE can be passed to humans as the so-called New-variant Creutzfeldt-Jakob Disease. Such issues command much of the current public attention about these otherwise rare neurologic disorders.
Examples of animal prion diseases include scrapie, which affects sheep and goats and takes its name from the tendency of affected animals to scrape against objects to relieve itching; transmissible mink encephalopathy, chronic wasting disease, which affects mule deer and elk; bovine spongiform encephalopathy; spongiform encephalopathy of exotic ruminants; and feline spongiform encephalopathy. Examples of currently identified human prion diseases include Kuru, Creutzfeldt-Jakob Disease, whether sporadic, familial, or iatrogenic, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker Syndrome, and New-variant Creutzfeldt-Jakob Disease.
The evidence of transmissibility of prion diseases between species has created fear. This is best evidenced by the destruction of cattle and bans on the importation of beef and cattle from Britain following an increased number of cases of mad cow disease. More information regarding aspects of prion diseases can be found at: www.jhu-prion.org and also at other internet sites.
A substantial amount of research has been carried out to determine how to inactivate prions from a range of materials. Researchers in general have found that it is a difficult task to identify successful conditions to achieve inactivation, and due to the severe conditions required, there has been little ability to correlate successful conditions in one composition to another. The most important reason for the inability to have a reasonable expectation of success in treating a previously untreated solution is that the conditions to treat the prion must be harsh (typically more extreme than those that are known to kill viruses), yet the conditions must be mild enough not to adversely affect the biological activity of the other components of the composition, if that material is to be used subsequently for a biological purpose, and is not merely being sterilized. This represents a difficult and sensitive balance.
For example, U.S. Pat. No. 5,756,678 to Shenoy, issued on May 26, 1998, describes that the inventors had discovered that it is possible to treat solutions of connective tissue material for the inactivation of prions in a manner that connective tissue molecules are not adversely affected by the inactivation treatment. The inventors found that treatment of the collagen with 0.1 to 0.35M NaOH inactivated the prions, but that the collagen was adversely affected unless they took the extra step of solubilizing the collagen prior to treatment. The inventors wrote:
From a prion deactivation perspective, it is preferable to treat a solution of collagen with sodium hydroxide rather than to treat precipitated fibers in a dispersion. The soluble collagen molecule and any beginning fibrils which are in solution are dissociated to permit maximum availability of any infectious agents which can reside in or be trapped with fiber structures. The collagen triple helix is too tightly wound (1.5 nm diameter) for viruses and prions (to the extent that they are known) to reside within the collagen molecule. Therefore, such virus or prion would be present either in the solution or absorbed onto the surface of a collagen molecule. In the soluble environment, where collagen fibers are dissociated into collagen molecules, there is no mass transfer barrier which requires the sodium hydroxide to diffuse through solids (assembled fibers) to reach the infectious agents on the surface of collagen molecules.
""678 patent, column 16, lines 20-35.
J. C. Darbord (Biomed and Pharmacother. 1999; 53: 34-8) described the ability of several procedures to sterilize medical laboratory materials that have come into contact with prion-infected tissue. Sterilizing conditions recommended by World Health Organization are a) incineration or quarantine; b) soaking in 1 N sodium hydroxide (1 h, 20xc2x0 C.); c) soaking in 17.5% bleach (1 h, 20xc2x0 C.) and d) steam sterilization in autoclave (134xc2x0 C.-138xc2x0 C., 18 min).
The first study of the distribution of the TSE prion during plasma fractionation found partitioning to occur predominantly into the initial precipitates obtained during the fractionation of murine plasma from animals infected with a human TSE, and from plasma prepared from human blood to which hamster-adapted scrapie 263K had been added. However, the distribution of TSE was not fully determined (Brown, et al., Transfusion, The distribution of infectivity in blood components and plasma derivatives in experimental models of TSE transfusion 1998:38:810-816).
Foster et al. (Transfusion, Microbiology and Plasma Fractions, Vox Sang, Studies on the Removal of Abnormal Prion Protein by Processes Used in the Manufacture of Human Plasma Products 2000; 78: 86-95), concluded that plasma ethanol fractionation processes used in the manufacture of albumin, immunoglobulin, factor-VIII concentrate, factor-IX concentrates, fibrinogen and thrombin all contain steps which can be capable of removing causative agents of human TSEs but that further studies are required.
Recommendations for Minimizing the Risk of Infection by Agents Causing Zoonoses and Other Animal Infections in the Manufacture of Medicinal Products, Federal Journal of Official Publications (BAnz., Germany), No. 164, p.6120 (1991), describes the sterilization of medical materials with a solution of 1N (1M) NaOH for one hour at 20xc2x0 C. for the purpose of inactivation of infectious agents. This treatment was recommended particularly for application to bovine spongiform encephalopathy (BSE) and materials of bovine origin.
Public Health Issues Related to Animal and Human Spongiform Encephalopathies: Memorandum from a WHO Meeting, Bulletin of the World Health Organization, 70(2): pp 183-190 (1992) recommended that medicinal products derived from bovine tissues be treated with NaOH, preferably 1M, for 1 hour at 20xc2x0 C. during the manufacturing process for removal or reduction of BSE infectivity.
Darwin R. Ernst and Richard E. Race, in xe2x80x9cComparative analysis of scrapie agent inactivation methodsxe2x80x9d Journal of Virological Methods, 41 (1993) 193-202, describe inactivation treatments for scrapie-infected hamster brain homogenate. Inactivation treatments utilizing autoclaving for various lengths of time either alone or in combination with different concentrations of sodium hydroxide, or an aqueous acid phenolic derivative, was disclosed. Although this paper indicates that treatment of suspensions of hamster brain using either 0.1N or 1.0N NaOH alone was carried out, no data are presented. D. M. Taylor et al., in xe2x80x9cDecontamination studies with the agents of bovine spongiform encephalopathy and scrapiexe2x80x9d, Arch. Virol. (1994) 139: 313-326, describe the use of sodium hydroxide to treat macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent; rodent brain infected with the 263K strain of scrapie agent; and, rodent brain infected with the ME7 strain of scrapie agent. The macerates were exposed for up to 120 minutes to 1.0M or 2.0M sodium hydroxide, but xe2x80x9cno procedure produced complete inactivation of all agents tested. Taylor et al. explained that the study was carried out due to inconsistencies in the data from various laboratories. They found that the data from the NaOH inactivation experiments demonstrated that none of the combinations of time (30 minutes up to 120 minutes) and molarity (1M and 2M) produced consistent inactivation of BSE and scrapie agents. Further, there was the unexplained finding in the NaOH experiments that with the 263K strain, with BSE, and possibly with ME7, two hours of exposure were less effective than exposure periods for 30 or 60 minutes.
In 1994 it was reported that homogenates of BSE-infected bovine brain exposed to less than or up to 120 minutes to solutions of sodium hypoclorite resulted in sterilization of the material, but that dichloroisocyanurate did not produce complete deactivation, nor did 1M or 2M sodium hydroxide treatment for up to 120 minutes. Taylor, et al., Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie Arch Virol (1994) Archives of Virology 139:313-326.
In reviewing the literature on attempts to inactivate prions, it is clear that the intention of much of the work has been to sterilize material, i.e., to destroy the prion without regard to the effect on the infected material, as opposed to finding conditions that destroy the prion while not adversely affecting the base infected material. This is seen from the severity of conditions used, i.e., autoclaving, treatment with hypochlorite (which oxidizes biological materials) and strong base (which can denature or degrade biological material).
Given the important biological uses of lipoproteins isolated from biological sources, and the risk of infection with prions, it is a goal of the present work to provide a process to reduce or eliminate prions from lipoprotein containing material.
It is an object of the present invention to provide a method to inactivate prions from a lipoprotein in a manner that does not substantially adversely affect the biological activity of the lipoprotein.
It is another object of the invention to provide a method to inactivate prions from an optionally-cholesterol carrying lipoprotein in a manner that does not substantially adversely affect the biological activity of the lipoprotein, wherein the lipoprotein is mammalian high density lipoprotein or low density lipoprotein.
It is still another object of the invention to provide a method to inactivate prions from an optionally-cholesterol carrying lipoprotein in a manner that does not substantially adversely affect the biological activity of the lipoprotein, wherein the lipoprotein is bovine lipoprotein.
It is another object of the invention to provide a method to inactivate prions from an optionally-cholesterol carrying lipoprotein composition that partially or completely sterilizes the composition.
It is another object of the invention to provide a method to inactivate prions from an optionally-cholesterol carrying lipoprotein composition that partially or completely sterilizes the composition, wherein the lipoprotein is mammalian high density lipoprotein or low density lipoprotein.
It is another object of the invention to provide a method to inactivate prions from an optionally-cholesterol carrying lipoprotein composition that partially or completely sterilizes the composition, wherein the lipoprotein is bovine high density lipoprotein or low density lipoprotein.
A process for treatment of a purified lipoprotein material to inactivate prions in a manner that does not substantially adversely affect the biological activity of the lipoprotein is provided that includes treating the lipoprotein prion composition with a solution of base at a pH of between 10 and 13 for a sufficient time to cause inactivation.
The term purified lipoprotein material refers to material (i) that can include any lipophilic compound that is typically carried through the plasma by apolipoproteins, including but not limited to cholesteryl esters, unesterified cholesterol, triglycerides, fatty acids and/or phospholipids; and (ii) that is in a higher state of purity than that found naturally in biological materials such as tissue or brain homogenate. In preferred embodiments, the purified lipoprotein material constitutes up to 60, 70, 80 or 90 percent or higher by weight of the material being treated. In one embodiment, the lipoprotein and cholesterol are in substantially pure form, i.e., the material being treated consists essentially of lipoprotein material.
In an alternative embodiment, purified lipoprotein material is treated with a base at a pH of above 8 or 9 to 13 inactivate prion for a period of time from initial contact up to 8 to 10 hours or more, preferably at approximately room temperature.
It was surprising to find that this treatment inactivates prions in the lipoprotein while not destroying or unduly affecting the sensitive biological material. It was not known prior to this work whether the lipoprotein would shield the prion from inactivation, or whether any condition could be found that inactivated the prion without also harming the lipoprotein. In one embodiment, the lipoprotein is treated as a somewhat dilute solution, to allow the base to easily penetrate the lipoprotein, enveloped by the more water soluble apolipoprotein.
Any suitable alkaline agent can be utilized to adjust the pH. According to one example, NaOH in a 1N solution was added to the lipoproteins to achieve an elevated pH of between 10 to about 13. The exposure to the elevated pH can include any exposure from the briefest possible exposure up to 8-10 hours. The lipoproteins can be exposed to an alkaline agent and the agent immediately neutralized. In such case, the pH is not maintained at the elevated pH, but rather adjusted to the elevated value and then immediately readjusted for minimal impact on the biological material. It appears that even such a brief exposure can help to reduce the TSE causing agent. Even though the pH exposure can be contact only, the exposure is typically at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. More typically, the pH is maintained at about 10 to about 13 for about 2 hours to about 8 hours. According to one embodiment, the solution is maintained at a pH of about 12 for about 8 hours. Longer periods of time can be utilized for the elevated pH exposure if deemed desirable and/or necessary. Those of ordinary skill in the art, once aware of the disclosure contained herein can determine acceptable pH levels and time periods without undue experimentation.
In another embodiment, any base can be used that does not denature or otherwise adversely affect the lipoprotein. For example, potassium hydroxide or other source of basic hydroxide ion that does not denature or otherwise adversely affect the lipoprotein can be used. Alternatively, the base can be an ammonium ion or amine, including mono or di(alkyl or alkanol)amine, or a carbonate or bicarbonate or mixture thereof, such as potassium or sodium carbonate or bicarbonate or a combination thereof. If the lipoprotein is to be used for a biological purpose, it is important that a base be used that will not leave a toxic residue.
Time and pH appear to be related, in that the higher the pH used, the shorter the amount of time that the infected material can or perhaps should be treated, to minimize the adverse affect on the lipoprotein material. The elevated pH exposure step can be carried out at any desired temperature, optimally between about 16xc2x0 C. and about 24xc2x0 C. According to one particular embodiment, the elevated pH exposure step was carried out at a temperature of room temperature, or about 20xc2x0 C. Temperature and time are related as are pH and time. For example, a higher temperature may be utilized for a shorter period of time, again to prevent an adverse affect on the treated lipoprotein material.
The lipoproteins treated by this process maintain their usefulness, for example, as a growth enhancement media supplement. Known processes do not disclose or suggest a process for producing a growth enhancement media supplement, wherein the process reduces, eliminates, or inactivates transmissible spongiform encephalopathy agent that can be present in portions of the raw materials used in the process to result in a final product that has little or no TSE agent present.
A typical but not necessary range of lipoprotein concentration in the treatment solution is between 10 and 3,500, and more particularly between 10 and 1,500 mg/dL. One particularly preferred range is between 50 and 500 mg/dL. After treatment with the base for a sufficient time to allow a desired degree of prion inactivation, the pH can be adjusted to neutral or another desired pH, using a pH-adjusting agent that does not adversely affect the biologic material.
In one embodiment a process for inactivating prions from a lipoprotein solution is provided, wherein the solution (other than contaminating prion) consists substantially or essentially of lipoprotein (optionally along with any lipophilic material carried by apolipoproteins through the plasma such as fatty acids, triglycerides, phospholipids or cholesterol), solvent, along with an insubstantial amount of other biological materials such as albumin. In one embodiment, the lipoprotein is (other than contaminating prion) substantially pure lipoprotein that may contain cholesterol. In a broader embodiment, the protein other than lipoprotein is present in less than 5, 4, 3, 2, 1, or 0.5% by weight.
In another embodiment, the solution contains at least approximately 0.1 to 8% by weight of optionally cholesterol bearing lipoprotein, and in particular up to approximately 0.1, 0.5, 1, 2, 4, 6, or 8% by weight of optionally cholesterol bearing lipoprotein.
In another embodiment the lipoprotein material consists substantially or essentially of HDL or LDL or combination thereof optionally in association with cholesterol, in an appropriate solvent. In a non limiting embodiment solvent can be, for example, water, saline, buffer, or any other aqueous solvent that does not adversely affect the biological properties of the material. Solutes that do not adversely or materially affect the biological properties of the material or the deactivation process can be included in the solution.
In a further embodiment, the lipoprotein material after prion deactivation is effective for use as a component of cell growth media.
In another embodiment, the treated lipoprotein is used for a purpose other than as nutrient in cell growth media, for example, as a source of lipoprotein for a host animal or organism in need thereof, for desired cholesterol transport, or for other biological purposes, including those associated with the presence of other lipophilic components such as fatty acids or phospholipids.
In another embodiment, prions are removed from a lipoprotein solution by contacting the solution with an adsorbant, preferably silica, which binds more tightly to the lipoprotein than to the prion. For example, the lipoprotein can be mixed with silica at a pH that does not cause the removal of the lipoprotein from the silica, typically between 6 and 8, and then the silica/lipoprotein particulate is separated from the prion-containing liquid by filtration. The lipoprotein is then removed from the silica using any appropriate method, for example, at an elevated pH. According to one embodiment, the recovery is carried out at a pH of about 10.5. According to another embodiment, the recovery is carried out by passing a high pH buffered solution through the lipoprotein-adsorbent complex until the lipoprotein is substantially removed from the adsorbent. After recovering the purified lipoproteins, the adsorbent can be discarded.
Still other objects and advantages of the present invention will become readily apparent by those skilled in the art from a review of the following detailed description. The detailed description describes preferred embodiments of the present invention, by way of illustration. As will be realized, the present invention is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is illustrative in nature and not meant to restrict the scope of the invention.
It has been discovered that prions can be inactivated from lipoprotein material in a manner that does not substantially adversely affect the biological activity of the lipoprotein, by treating the lipoprotein with a solution of sodium hydroxide or other base to a pH of between 10 (or even pH 8 or 9) and 13 for a period of time from contact to 8 to 10 hours, preferably at approximately room temperature. It was surprising to find that this treatment inactivates prions in the lipoprotein material while not destroying or unduly affecting the sensitive biological material. It was not known prior to this work whether the lipoprotein material would shield the prion from inactivation, or whether any condition could be found that inactivated the prion without also harming the lipoprotein. In one embodiment, the lipoprotein material is treated as a somewhat dilute solution, to allow the base to easily penetrate the lipoprotein, enveloped by the more water soluble apolipoprotein. An example of the range of lipoprotein concentration is between 10 and 3,500 mg/dL, and one particularly preferred range is between 50 and 200 mg/dL. After treatment with the base for a sufficient time to allow a desired degree of prion inactivation, the pH can be adjusted to neutral or another desired pH, using a pH-adjusting agent that does not adversely affect the biologic material.
Any base can be used that does not denature or otherwise adversely affect the lipoprotein. For example, sodium hydroxide or potassium hydroxide or other source of basic hydroxide ion that does not denature or otherwise adversely affect the lipoprotein can be used. Alternatively, the base can be an ammonium ion or amine, including mono or di(alkyl or alkanol)amine, or a carbonate or bicarbonate or mixture thereof, such as potassium or sodium carbonate or bicarbonate or a combination thereof. If the lipoprotein is to be used for a biological purpose, it is important that a base be used that will not leave a toxic residue.
The lipoproteins that are treated according to this invention, for example, can be that taken from any TSE infected animal blood plasma or serum fraction, and in particular, from a mammal, including that from bovine, horse, sheep, pig or human plasma or serum or fraction thereof, such as fibrinogen-poor plasma, Cohn Fraction I supernatant, ammonium sulfate supernatant rich in lipoprotein and other fractions. Typical source materials are bovine serum and bovine plasma.
In one embodiment a process for inactivating prions from a lipoprotein solution is provided, wherein the solution consists substantially or essentially of lipoprotein material and solvent, along with an insubstantial amount of other biological materials, for example, albumin.
In another embodiment, the solution contains at least approximately 0.1 to 8% by weight of lipoprotein material, and in particular up to approximately 0.1, 0.5, 1, 2, 4, 6, or 8% by weight of lipoprotein material.
In another embodiment the solution consists substantially or essentially of HDL or LDL or combination thereof optionally in association with cholesterol, in an appropriate solvent. In a non-limiting embodiment solvent can be, for example, water, saline, buffer or any other aqueous based that does not adversely affect the biological properties of the material. Solutes that do not adversely affect the biological properties of the material or the deactivation process can be included in a solution.
In a further embodiment, the lipoprotein material after prion inactivation is used as a component of cell growth media.
In another embodiment, prions are removed from a lipoprotein material solution by contacting the solution with an adsorbant, preferably silica, which binds more tightly to the lipoprotein than to the prion. For example, the lipoprotein can be mixed with silica at a pH that does not cause the removal of the lipoprotein from the silica, typically between 6 and 8, and then the silica/lipoprotein particulate is separated from the prion-containing liquid by filtration. The lipoprotein is then removed from the silica using any appropriate method, for example, at an elevated pH. According to one embodiment, the recovery is carried out at a pH of about 10-11. According to another embodiment, the recovery is carried out by passing a high pH buffered solution through the lipoprotein-adsorbent complex until the lipoprotein is substantially removed from the adsorbent. After recovering the purified lipoproteins, the adsorbent can be discarded.
The following provides illustrations of the process according to the present invention. The general discussion is followed by examples of two specific embodiments. The effectiveness of the present invention in eliminating transmissible spongiform encephalopathy agents is further discussed below.